The goal of cancer therapy is to achieve a degree of selectivity that spares normal cells and destroys all malignant ones, since even a small number of remaining malignant cells can lead to recurrence, metastasis, and death. A two-component or binary system comprised of constituents that alone are nonlethal and largely confined to malignant cells, and which when combined are lethal to the neoplastic cells yet innocuous to normal cells is an ideal modality. One advantage of this type of binary system is that each component can be manipulated independently to maximize selectivity.
Boron neutron capture therapy (BNCT, see FIG. 1) is a binary system which combines two separately nonlethal constituents, a radio sensitizing compound that contains a stable boron-10(.sup.10 B) isotope, and nonionizing neutron radiation. When boron-10 is irradiated with neutrons, a nuclear reaction occurs that yields helium nuclei (.alpha.-particle), lithium nuclei, and about 100 million times more energy than the initial irradiated energy. The generated radiation destroys malignant cells containing the boron compound. Selectivity is achieved through the use of compounds which accumulate primarily in malignant cells and/or by aiming the neutron beam at the tumor mass which contains the boron carrier.
The major obstacles in BNCT are: (1) the achievement of a sufficiently high intracellular boron concentration and (2) selectivity toward tumor cells. Although attempts to develop tumor-selective boron compounds date back to the 1960s and despite extensive studies, the problem of selective delivery of boron carriers to tumor cells remains.
Many classes of compounds have been synthesized for BNCT. For example, see Barth, R. F.; Soloway, A. H.; Fairchild, R. G.; Brugger, R. M. Cancer 1992, 70, 2995-3008; Fairchild, R. G.; Kahl, S. B.; Laster, B. H.; Kalef-Ezra, J.; Popenoe, E. A. Cancer Res. 1990, 50, 4860-4865; and Zamenhof, R. G.; Kalend, A. M.; and Bloomer, W. D. J Natl Cancer Inst 1992, 84, 1290-1291.
The first boron-containing nucleoside, 5-dihydroxyboryl-2'-deoxyuridine, was synthesized by Schinazi and Prusoff in 1978. Schinazi, R. F., Prusoff, W. H. Tetrahedron Lett 1978, 4981-4984; and Schinazi, R. F.; Prusoff, W. H. J. Org Chem 1985, 50, 841-847. Sood et al. have reported the synthesis of a series of cyanoborane adducts of 2'-deoxynucleosides, specifically 2'-deoxyguanosine-N.sup.7 -cyanoborane, 2'-deoxyinosine-N.sup.7 -cyanoborane, 2'-deoxyadenosine-N.sup.1 -cyanoborane, and 2'-deoxycytidine-N.sup.3 -cynoborane. Sood, A.; Spielvogel, B. F.; Shaw, B. R. J Am Chem Soc 1989, 111, 9234-9235.
Sood et al. have also reported the synthesis of oligonucleotides with a boronated internucleotide backbone, in the form of boranophosphates and boranophosphate methyl esters. The borane (BH.sub.3) group in these boronated oligonucleotides is isoelectronic and isostructural with normal O-oligonucleotides and oligonucleotide methylphosphonates. Sood, A.; Shaw, B. R.; Spielvogel, B. F. J Am Chem Soc 1990, 112, 9000-9001. The Sood compounds in general have a low boron content and some have lower than desired lipophilicity.
U.S. Pat. No. 5,130,302 to Spielvogel, et al., discloses a novel class of boronated nucleosides, nucleotides and oligonucleotides for use as antineoplastic, antiinflammatory, and antihypertensive agents. The nucleosides, nucleotides and oligonucleotides are covalently attached to either BH.sub.2 CN, BH.sub.3, or BH.sub.2 CO.sub.2 R moieties, wherein R is C.sub.1 to C.sub.18 alkyl.
A number of carboranyl pyrimidines have been prepared for use in boron neutron capture therapy. Examples of carboranyl pyrimidines include 5-(3-o-carboranylproyl-6-methyl-2-thiouracil (compound A) (Wilson, J. G. Pigment Cell Res 1989, 2, 297-303), 2,4-dichloro-5-(1-o-carboranylmethyl)-6-methylpyrimidine; (compound B) (Reynolds, R. C.; Trask, T. W.; Sedwick, W. D. J Org Chem 1991, 56, 2391-2395); and 5-carboranyluracil (compound C) (Goudgaon, N. M.; El-Kattan, Y.; Fulcrand, G.; Liotta, D. C.; Schinazi, R. F. IMEBORON VIII, Knoxville, Tenn.; p 72, 1993).
Purine and pyrimidine nucleosides that contain a carboranyl group attached to the purine or pyrimidine base have also been reported. Yamamoto, Y.; Seko, T.; Nakamura, H. Heteroatom Chem 1992, 3, 239-244; and Schinazi, R. F.; Goudgaon, N. M.; Soria, J.; Liotta, D. C. 5th International Symposium on Neutron Capture Therapy, Columbus, Ohio; p 11, 1992; Schinazi, R. F.; Goudgaon, N.; Soria, J.; Liotta, D. C. Tenth International Roundtable: Nucleosides and Nucleotides, Park City, Utah; p 28, 1992. These compounds are lipophilic and some are readily phosphorylated by cellular kinases, and in certain cells can incorporate into DNA as analogues of natural 2'-deoxypyrimidine nucleosides. Examples include 5-carboranyl-2'-deoxyuridine (compound D, CDU), 5-carboranyluridine (compound E, CU), 5-(1-hydroxymethyl)carboranyluridine, and 5-(1-hydroxymethyl)carboranyluridine (compound F, HMCU). ##STR1## ##STR2##
PCT WO 93/17028 filed by Raymond F. Schinazi and Dennis C. Liotta discloses a number of synthetic nucleosides that contain a carboranyl moiety covalently attached to a purine or pyrimidine base, wherein the sugar moiety optionally contains a second heteroatom in the 3'-position of the ring. Preferred compounds are 2-hydroxymethyl-5-(5-carboranylcytosin-1-yl)-1,3-oxathiolane (compound G) and 2-hydroxymethyl-5-(5-carboranyluridin-1-yl)-1,3-oxathiolane (compound H).
Powell, et al., recently reported the synthesis of oligonucleotides that contain 3',5'-nido-o-carboranyl-phosphoramidate linkages (compound I). While the oligonucleotide could reportedly localize in the cell nucleus, the boron moiety is acid labile because it is linked to the phosphorus atom through an amide-type bond.
The requirements for efficient BNCT with oligonucleotides, which include cell selectivity (ability to accumulate preferentially in diseased cells), stability of the chemotherapeutic agent in vivo (resistance against digestion by cellular nucleases and chemical stability), and transportability (ability of the chemotherapeutic agent to pass easily through cellular membranes), are very similar to the requirements for Antisense Oligonucleotide Technology (AOT), another recently developed therapy for cancer as well as other diseases. Uhlmann, "Antisense Oligonucleotides: A New Therapeutic Approach" Chemical Reviews, 90(4), June 1990. The compounds should also be relativel non-toxic. Antisense technology refers in general to the modulation of gene expression through a process wherein a synthetic oligonucleotide is hybridized to a complementary nucleic acid sequence to inhibit transcription or replication (if the target sequence is DNA), inhibit translation (if the target sequence is RNA) or to inhibit processing (if the target sequence is pre-RNA). A wide variety of cellular activities can be modulated using this technique. A simple example is the inhibition of protein biosynthesis by an antisense oligonucleotide bound to mRNA. In another embodiment, a synthetic oligonucleotide is hybridized to a specific gene sequence in double stranded DNA, forming a triple stranded complex (triplex) that inhibits the expression of that gene sequence. Antisense oligonucleotides can be also used to activate gene expression indirectly by suppressing the biosynthesis of a natural repressor or directly by reducing termination of transcription. AOT can be used to inhibit the expression of pathogenic genes, for example, those that facilitate the replication of viruses, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), and herpesviruses, and cancers, particularly solid tumor masses such as gliomas, breast cancer, and melanomas.
While progress has been made in the areas of both BNCT and AOT, none of the synthetic oligonucleotides prepared to date exhibit the optimal combination of cell selectivity, stability in vivo, and ability to pass easily through cellular membranes (transportability).
Therefore, it is an object of the present invention to provide a new class of synthetic oligonucleotides for use in BCNT, AOT, or both, that exhibit a desired profile of cell selectivity, stability in vivo, and ability to pass easily through cellular membranes.
It is another object of the present invention to provide new methods for the preparation of boron-containing nucleosides and oligonucleotides.